Cytotoxic effects of the dietary flavones chrysin and apigenin in a normal trout liver cell line.

Many flavonoids have been shown to possess prooxidant properties, capable of causing oxidative stress, especially at larger doses. Here, we examined the potential cell toxicity caused by exposure to the hydroxylated flavones chrysin, apigenin, luteolin and quercetin in comparison to the methylated flavones 5,7-dimethoxyflavone and 3',4'-dimethoxyflavone in normal Rainbow trout hepatocytes. The hydroxylated flavones, especially chrysin, demonstrated cell toxicity and inhibition of DNA synthesis at very low (2 microM) concentrations. The cytotoxicity of chrysin may partially be due to its metabolism by myeloperoxidase, which was shown to be present in these normal trout liver cells (164pmol/(min mg protein)). In contrast, methylated flavones showed no significant metabolism by myeloperoxidase and no signs of toxicity, even at much higher concentrations. These results may be useful for further investigations of cytotoxicity of dietary flavonoids.

Methylation protects dietary flavonoids from rapid hepatic metabolism.

The metabolic stability of two potential cancer chemopreventive flavones, i.e. 5,7-dimethoxyflavone (5,7-DMF) and 3',4'-dimethoxyflavone (3',4'-DMF), compared with the non-methylated flavone galangin (3,5,7-trihydroxyflavone), was investigated in human hepatic preparations. Galangin, as expected, was extensively metabolized mainly by glucuronidation in human liver S9 fractions in the presence of appropriate co-factors. In contrast, 5,7-DMF and 3',4'-DMF were metabolically highly stable with only a small fraction of 3',4'-DMF undergoing oxidation. Consistent with the S9 fraction results, galangin was almost completely depleted after 2-h incubations in freshly plated hepatocytes. The hepatocytes also showed some metabolism of 3',4'-DMF, but virtually none of 5,7-DMF. In human liver microsomes, 5,7-DMF was more metabolically stable than 3',4'-DMF. The observations present a new strategy for examining the metabolic stability of dietary flavonoids and suggest that methylated flavonoids may have a high oral bioavailability compared with their non-methylated forms, which will make them more likely to be useful as cancer chemoprotectants.

S-adenosyl-L-methionine: transcellular transport and uptake by Caco-2 cells and hepatocytes.

S-adenosyl-L-methionine (SAMe) is an endogenous molecule that is known to be protective against hepatotoxic injury. Although oral SAMe appears to be absorbed across the intestinal mucosa, its systemic bioavailability is low. The reason for this is unknown. Using the Caco-2 cell culture model for enterocyte absorption, we determined the mode by which SAMe is transported across this cell monolayer. We also determined the extent it is taken up by both Caco-2 cells and hepatocytes. In Caco-2 cells transport was observed in both apical to basolateral and basolateral to apical directions. The apparent permeability coefficients (Papp) appeared to be concentration independent and were similar in both directions (0.7x10(-6) and 0.6x10(-6) cm s-1, respectively), i.e. identical to that of the paracellular transport marker mannitol (0.9x10(-6) and 0.7x10(-6) cm s-1). This mode of transport was supported by a four-fold increase in the Papp for SAMe transport in Ca++-free buffer. Cellular uptake of SAMe was examined in both Caco-2 cells and cultured rat hepatocytes. Uptake by hepatocytes was not saturable in a concentration range of 0.001-100 microM. Accumulation by both cell types was very low, with a cell:medium ratio at equilibrium of only 0.2-0.5. This low cell accumulation supports the finding of paracellular transport as the only mode of cell membrane transport. Increased hepatocellular protection for SAMe may be accomplished by converting SAMe to a more lipid-soluble prodrug.

Familial and sporadic inflammatory bowel disease: comparison of clinical features and serological markers in a genetically homogeneous population.

BACKGROUND: The familial occurrence of inflammatory bowel disease (IBD) and the clinical features of familial and sporadic IBD in the genetically homogeneous Finnish population are evaluated. METHODS: 257 patients with Crohn disease (CD) and 436 with ulcerative colitis (UC) participated in the study. They were asked whether IBD was present (familial IBD) or absent (sporadic IBD) in their first-degree relatives. Data on the clinical course of the disease were collected from the patient records. Antibodies to Saccharomyces cerevisiae (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) were determined from serum samples. RESULTS: Affected first-degree relatives were found in 15.6% of patients with CD and in 13.8% of patients with UC. In familial cases CD was more often located in the ileum (38% versus 21%) and less often in the ileocolon (35% versus 50%) (P< 0.05) than in sporadic cases. A greater percentage of CD patients than UC patients were smokers (47% versus 13%; P < 0.01). An elevated level of IgA and/or IgG antibodies for ASCA was found more often in CD patients than in UC patients (59% versus 14%; P < 0.01), while pANCA were found more often in UC than in CD patients (48% versus 12%; P < 0.01). The combination of pANCA-ASCA+ yielded a sensitivity, specificity and positive predictive value of 48%, 92% and 90%, respectively, for CD, and the combination of pANCA + ASCA- of 55%, 94% and 90%, respectively, for UC. CONCLUSIONS: The percentage of familial IBD cases in Finland is comparable to that reported elsewhere in Europe. No important clinical differences between patients with familial and sporadic forms of the disease were found. ASCA is associated with both familial and sporadic CD and pANCA with UC, but low sensitivity diminishes their value as a serological marker of IBD or as a differential diagnostic test between CD and UC.

Transport and metabolism of the tea flavonoid (-)-epicatechin by the human intestinal cell line Caco-2.

PURPOSE: Tea flavonoids, including (-)-epicatechin (EC), have been suggested to have chemopreventive properties in cancer. However, there is limited knowledge of the oral bioavailability of these dietary compounds. The purpose of this study was to gain a better understanding of the absorption of EC. METHODS: The intestinal epithelial membrane transport of EC was examined using the monolayer of the human Caco-2 cell line grown in Transwells, a common model of intestinal absorption. EC and its metabolites were measured by high performance liquid chromatography with diode array detection. RESULTS: EC showed no apical to basolateral absorption at concentrations ranging from 5 to 50 microM. In contrast, EC demonstrated basolateral to apical efflux with a Papp value of 0.67 +/- 0.05 x l0(-6) cm/sec, i.e., slightly higher than for mannitol, 0.50 +/- 0.30 x 10(-6) cm/ sec, a paracellular transport marker. There was a 50% reduction in the efflux of EC in the presence of 50 microM MK-571, a competitive inhibitor of the MRP2 transporter expressed in the apical membrane of Caco-2 cells. Most important. the presence of 50 microM MK-571 resulted in clearly measurable apical to basolateral absorption of EC with a Papp of 0.31 +/- 0.06 x 10(-6) cm/sec. Two polar metabolites, M1 and M2, were formed from EC, both of which appeared exclusively on the apical side. MK-571 (50 microM) dramatically inhibited the transport for both of these metabolites. Incubations with inorganic 35SO4(2-) and hydrolysis by aryl sulfatase strongly suggested that these metabolites were sulfate conjugates. CONCLUSIONS: These results suggest an important role for the multispecific organic anion transporter MRP2 in the bioavailability of EC and possibly other tea flavonoids.

Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in Caco-2 cells--potential role in carcinogen bioinactivation.

PURPOSE: Dietary flavonoids, present in fruits, vegetables and beverages have been demonstrated to be protective in cancer. Recently, we showed that the flavonoid chrysin induced UDP-glucuronosyltransferase (UGT) activity and expression in the human intestinal cell line Caco-2. In the present study, we determined the specific UGT isoform(s) induced and whether this induction facilitates glucuronidation and potential detoxification of the colon carcinogen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). METHODS: The induction was studied by immunoblot analysis with UGT isoform-specific antibodies, by Northern blot analysis and using quercetin as an isoform-specific catalytic probe. Glucuronidation of N-hydroxy-PhIP was characterized using both recombinant UGTs and control and chrysin-treated microsomes. RESULTS: Western blot analysis showed that pretreatment of Caco-2 cells with 25 microM chrysin induced UGT1A1 without affecting the expression of UGTs 1A6, 1A9 and 2B7. Northern blot analysis showed markedly increased expression of UGT1AI mRNA after chrysin treatment. Similarly, glucuronidation of quercetin was greatly increased in a UGT1A1-specific way. The induction of UGT1A1 in the Caco-2 cells resulted in a 10-fold increase in the glucuronidation of N-hydroxy-PhIP. CONCLUSION: Dietary flavonoid-mediated induction of intestinal UGT1A1 may be important for the glucuronidation and detoxification of colon and other carcinogens as well as for the presystemic metabolism of therapeutic drugs.

Quercetin and resveratrol potently reduce estrogen sulfotransferase activity in normal human mammary epithelial cells.

Estrogen sulfotransferase (EST) is the sole sulfotransferase expressed in normal human breast epithelial cells and has an important function in determining free estrogen hormone levels in these cells. In the present study we examined the inhibitory effect of the dietary polyphenols quercetin and resveratrol on EST activity, i.e. 17beta-estradiol (E2) sulfation. Both the compounds potently inhibited recombinant human EST in a competitive fashion with K(i) values of about 1 microM. In fact, both polyphenols could serve as substrates for EST. In order to extend the studies to more physiologically relevant conditions, we examined whether inhibition of EST also occurred in the intact cultured human mammary epithelial (HME) cells. The mean baseline EST activity (E2 sulfate formation) in the HME cells was 4.4 pmol/h per mg protein. The IC(50) for resveratrol was very similar to that for recombinant EST, i.e. about 1 microM. Surprisingly, quercetin was 10 times more potent in the HME cells with an IC(50) of about 0.1 microM, a concentration that should be possible to achieve from the normal dietary content of this flavonoid.

Quercetin glucosides are completely hydrolyzed in ileostomy patients before absorption.

Flavonoids, dietary components in vegetables, fruits and beverages, may protect against coronary heart disease, stroke and cancer. However, the bioavailability of these compounds is questionable. A previous study in ileostomy patients of the most abundant flavonoid, quercetin, suggested a 52% absorption of its major dietary forms, monoglucoside (QMG) and diglucoside (QDG), from an onion meal. However, this was based on indirect measurements after acid hydrolysis. Because human intestinal Caco-2 cell monolayers showed minimal absorption of the glucosides, we repeated the study in ileostomy patients, using molecularly specific analytical methodology for the intact glucosides and quercetin. The onion meal had high concentrations of both QMG and QDG with only trace amounts of quercetin. The intake of QMG and QDG in four patients ranged from 10.9 to 51.6 mg. No QMG or QDG was detected in the ileostomy fluid. In contrast, the amounts of the aglycone quercetin were substantial, 2.9-11.3 mg. This corresponded to 19.5-35.2% of total quercetin glucosides ingested, implying absorption of 64.5-80.7%. These findings suggest a different interpretation than that from the previous study, i.e., that both QMG and QDG are efficiently hydrolyzed in the small intestine by beta-glucosidases to quercetin, most of which is then absorbed.

Induction of UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line hep G2.

The UDP-glucuronosyltransferases (UGTs) have long been known to be inducible by various chemicals, including drugs, although the extent of induction in general has been modest. In the present study, we determined the ability of the dietary flavonoid chrysin to induce UGT activity, protein and mRNA. When pretreating human hepatoma Hep G2 cells with 25 microM chrysin, the glucuronidation of chrysin itself increased 4.2-fold when measured in the intact cell and 14-fold in the cell homogenate, i.e., autoinduction. Microsomes from chrysin-treated cells probed with specific antibodies in Western analyses showed marked induction of the UGT1A family of proteins. Isoform-specific induction of the important hepatic UGT1A1 protein was observed but not of UGT1A6 or UGT2B7. The strong induction of UGT1A1 was confirmed by Northern analyses of total RNA as well as mRNA, using a specific probe. UGT1A1 message as well as protein was detectable also in untreated Hep G2 cells. In catalytic activity assays with recombinant UGT1A1, 1A4, 1A6 and 1A9, chrysin was found to be a high affinity substrate for UGT1A1 (K(m) 0.35 microM). Catalytic activity was also found for UGT1A9 and 1A6 but not for 1A4. Further studies demonstrated a 20-fold induction of the glucuronidation of bilirubin by the chrysin-treated cells and a 7. 9-fold induction of the glucuronidation of the oral contraceptive drug ethinylestradiol, two of the best known and specific UGT1A1 substrates, demonstrating the potential importance of this induction. In view of these findings, it will be important to extend these studies to other dietary flavonoids.

Sulphonation of N-hydroxy-2-acetylaminofluorene by human dehydroepiandrosterone sulphotransferase.

1. The aim was to determine which human recombinant sulphotransferase (ST) isoform(s) were responsible for the sulphonation and, thus, potential further bioactivation of the classical hepatic procarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). 2. N-OH-2AAF was incubated together with the cosubstrate 3'-phosphoadenosine-5'-phosphosulphate (PAPS) and either human liver cytosol or recombinant P-form phenolsulphotransferase (P-PST), M-form PST, dehydroepiandrosterone-ST (DHEA-ST) or oestrogen ST (EST). Formation of 3'-phosphoadenosine-5'-phosphate (PAP) from PAPS, measured by HPLC, was used as the assay for determination of sulphoconjugation rates. 3. The liver cytosol produced a 100% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline levels (p < 0.01), corresponding to a rate of 19 pmol/min/mg protein. Recombinant P-PST, however, was without effect. This is in contrast to previous suggestions using crude enzyme preparations. Like P-PST, recombinant M-PST and EST did not sulphonate N-OH-2AAF. On the other hand, recombinant DHEA-ST produced a 161% increase in PAP formation in the presence of 200 microM N-OH-2AAF as compared with baseline values (p < 0.001). 4. Kinetic studies of N-OH-2AAF sulphonation by DHEA-ST and human liver cytosol gave similar apparent Kms. Interestingly, the Vmax for N-OH-2AAF sulphonation by DHEA-ST was very similar to that of DHEA, the natural substrate for DHEA-ST. 5. This is the first paper to demonstrate the involvement of the human DHEA-ST in the sulphonation of an N-hydroxylated aromatic amide carcinogen.

Induction of UDP-glucuronosyltransferase by the flavonoids chrysin and quercetin in Caco-2 cells.

PURPOSE: Dietary flavonoids have been reported to be potent inhibitors of drug metabolizing enzymes. In the present study we examined the inducing effect of three of these compounds, chrysin, quercetin and genistein, on UDP-glucuronosyltransferase (UGT) in the human intestinal cell line Caco-2. METHODS: The induction of UGT by flavonoid pretreatment was studied both in the intact cells and cell homogenates, measured as the glucuronidation of chrysin, and by immunoblot analysis of the UGT 1A protein. RESULTS: Exposure of Caco-2 cells to 50 microM chrysin resulted in a 3.8-fold increase in chrysin glucuronidation in intact cells (p < 0.0001) with a 38% decrease in sulfation (p < 0.01). In the cell homogenate the induction was much larger, 14-fold. The induction was slow to develop with maximum induction after 3-4 days. Interestingly, the isoflavonoid genistein was without effect. Immunoblot analysis of Caco-2 cell microsomes with a UGT1A subfamily-selective antibody showed a markedly increased band at about 59 kDa, consistent with induction of one or more UGT1A isoforms. A 5-week exposure of Caco-2 cells to low concentrations (10 microM) of chrysin or quercetin also showed markedly increased glucuronidation activity. CONCLUSIONS: Diet-mediated induction of intestinal UGT may be important for the bioavailability of carcinogens and other toxic chemicals as well as therapeutic drugs.

Transport of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) across the human intestinal Caco-2 cell monolayer: role of efflux pumps.

Cooked-food mutagens formed when frying meat have been suggested to contribute to the etiology of colon, breast and prostate cancer. The most prevalent of these mutagens is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which after absorption is bioactivated by both phase I and phase II enzymes. Although available data suggest absorption of PhIP in humans, the extent and mechanism of absorption are unknown. In the present study we examined the transport of [(3)H]PhIP through the human Caco-2 intestinal epithelial cell monolayer, a well-accepted model of human intestinal absorption. The influx, or absorption, was extensive and linear for 2 h and up to a PhIP concentration of 5 microM. Still, the basolateral to apical efflux [apparent permeability coefficient (P(app)) 54.2 +/- 0.7x10(-6) cm/s, mean +/- SEM, n = 24] was 3.6 times greater than the apical to basolateral influx (P(app) 15.1 +/- 0.6x10(-6) cm/s, n = 21, P < 0.0001). Equilibrium exchange experiments demonstrated the efflux to be a true active process. Preincubations with verapamil, an inhibitor of P-glycoprotein-mediated transport, or MK-571, an inhibitor of multidrug resistance-associated protein-mediated transport, stimulated influx and reduced efflux of PhIP, suggesting that PhIP is a substrate for both of these transporters. These findings should be considered when determining exposure to the cooked food mutagens.

The influence of plasma binding on absorption/exsorption in the Caco-2 model of human intestinal absorption.

The Caco-2 cell monolayer has become an increasingly useful in-vitro model of human intestinal absorption. In this study we have determined the effect of plasma on the basolateral side on the absorption as well as exsorption of several drugs that are highly bound to plasma proteins. The drugs used included propranolol and quercetin, which both use the transcellular route of absorption, and taxol and oestradiol 17 beta-D-glucuronide, which are thought to undergo efflux by P-glycoprotein and the multidrug resistance protein MRP, respectively. All experiments were carried out under sink conditions to mimic normal absorption. It was necessary to use heparin anticoagulation for generation of the plasma, as EDTA was found to make the monolayers very leaky. The apparent permeability (P(app)) values for absorption were 1.54 x 10(-6) cm s(-1) for oestradiol 17 beta-D-glucuronide, 3.33 x 10(-6) cm s(-1) for taxol, 20.8 x 10(-6) cm s (-1) for quercetin, and 35.3 x 10(-6) cm s(-1) for propranolol. For these four compounds, plasma on the basolateral side had no influence on absorption. However, plasma on the basolateral side significantly reduced the efflux of oestradiol 17 beta-D-glucuronide by 66%, taxol by 75%, propranolol by 82%, and quercetin by 94%. Failure to consider the effect of plasma binding can result in an overestimate of basolateral to apical efflux and result in misleading net flux calculations.

Phase I and pharmacologic study of a 3-hour infusion of paclitaxel followed by cisplatinum and 5-fluorouracil in patients with advanced solid tumors.

A Phase I and pharmacological study of paclitaxel administered as an outpatient, 3-h i.v. infusion just before a 5-day regimen of daily cisplatinum (CP) and a continuous infusion of 5-fluorouracil (5-FU) was performed in patients with advanced solid tumors. A secondary objective was to determine the objective response rate to this regimen. Forty-two patients were enrolled and were evaluable for toxicities. Eighteen patients were previously untreated, whereas the rest had received prior treatment with radiation (J. H. Schiller et al., J. Clin. Oncol., 12: 241-248, 1994), chemotherapy (M. J. Kennedy et al., Clin. Cancer Res., 4: 349-356, 1998), or both modalities (J. H. Schiller et al., J. Clin. Oncol., 12: 241-248, 1994). The paclitaxel dose was escalated from 100-135-170-200-225 to 250 mg/m2, whereas i.v. 5-FU and CP doses were fixed at 1.0 g/m2/day continuous infusion and 20 mg/m2/day, respectively, daily for 5 days. Granulocyte colony-stimulating factor (G-CSF; 5 microg/kg/day) was administered s.c. from day 6, routinely after 250 mg/m2 dose of paclitaxel or after a lower dose of paclitaxel if ANC <500/microl or febrile neutropenia was observed. Patients were treated every 28 days. Plasma and urine samples were collected to determine the pharmacokinetics of paclitaxel. In previously untreated patients, the maximally tolerated dose of paclitaxel in the drug regimen was determined to be 170 mg/m2 without and 250 mg/m2 with G-CSF support. At the higher dose level, mucositis and thrombocytopenia were dose-limiting. In previously treated patients, these toxicities were observed at all dose levels of paclitaxel > or =135 mg/m2. With increasing doses of paclitaxel, a disproportionate increase in the peak concentrations, as well as the area under plasma concentration time-curve, was seen. This nonlinearity was due to saturable total body clearance and volume of distribution of paclitaxel (P < 0.001). The apparent plasma elimination half-life was unaffected by the dose of paclitaxel. CP and 5-FU had no apparent effect on the metabolism of paclitaxel. Among 32 patients evaluable for response, 22 demonstrated an objective response, including five complete remissions. Therefore, a regimen of 3-h infusion of 250 mg/m2 paclitaxel before CP and FU is tolerable with G-CSF (as above) support in previously untreated patients. The regimen also seems to be highly active against breast and esophageal cancers.

Transport of genistein-7-glucoside by human intestinal CACO-2 cells: potential role for MRP2.

This study demonstrated rapid apical to basolateral absorption of genistein across Caco-2 cell monolayers with a P(app) of 20.0+/-0.8 x 10(-6)cm/sec. In contrast, genistein's main dietary source, genistein-7-glucoside (genistin), was not absorbed. In fact, genistin demonstrated basolateral to apical efflux of 1.28+/-0.10 x 10(-6) cm/sec, which exceeded the flux of the paracellular transport marker mannitol by 6-fold. Although genistin hydrolysis to genistein occurred in the preparation, it did not contribute significantly to these observations. In experiments trying to define the efflux mechanism for genistin, MK-571 reduced the efflux by 87% to 0.19+/-0.02 x 10(-6) cm/sec, implying transport by the polarized efflux pump MRP2.

Fate of the flavonoid quercetin in human cell lines: chemical instability and metabolism.

Although cell cultures are increasingly being used as models for studying the biological actions of flavonoids, no information on the fate, such as uptake and metabolism, exists for these natural products in these models. This study examined the elimination of quercetin, one of the most abundant flavonoids, from the cultured human hepatocarcinoma cell line Hep G2 using [14C]-labelled compound with HPLC and LC/MS for structure characterization. These cells showed a 9.6-fold accumulation of quercetin and the formation of an O-methylated metabolite, isorhamnetin. However, a rapid elimination of quercetin, with no unchanged compound present beyond 8 h, was mainly due to oxidative degradation. The initial intermediate reaction appears to involve peroxidation, leading to a dioxetan, as evidenced by a 32-amu increase in the molecular ion by LC/MS. Subsequently, opening of the C-ring leads to the formation of carboxylic acids, the major one identified in this study as protocatechuic acid. A separate reaction results in a polymeric quercetin product which is highly retained on a reversed-phase C18 HPLC column. It is postulated that these degradative and metabolic changes contribute to the multiple biological actions reported for quercetin, using cell culture models. Interestingly, part of the degradative pathway could be inhibited by including nontoxic concentrations of EDTA in the cell culture medium.

Extensive metabolism of the flavonoid chrysin by human Caco-2 and Hep G2 cells.

1. Chrysin is one of many bioflavonoids with chemopreventive properties in cardiovascular disease and cancer. In an effort better to understand factors that may affect the oral bioavailability of the bioflavonoids from dietary sources, the metabolism of chrysin by cultured intestinal Caco-2 cells and hepatic Hep G2 cells was studied, together modelling human presystemic metabolism. 2. At concentrations that may be achieved in the diet, chrysin was extensively metabolized to two conjugated metabolites, M1 and M2, with no CYP-mediated oxidation. M1 was identified as a glucuronide, and M2 as a sulphate conjugate by LC/MS and other spectroscopic and biochemical techniques. Sulphate conjugation occurred at a rate twice that of glucuronic acid conjugation in both cell types. 3. M1 was catalyzed by UGT1A6 with a Km = 12 microM. M2 was catalyzed both by M- and P-form phenolsulphotransferases (SULT 1A3 and SULT 1A1) with very low Km of 3.1 and 0.05 microM respectively. 4. Pretreatment with 3-methylcholanthrene, interestingly, did not result in oxidation of chrysin but rather in increased glucuronidation. 5. Also, M1 and M2 were the only metabolites formed from chrysin in fresh rat hepatocytes. The metabolism of another flavonoid, apigenin, was very similar to that of chrysin. 6. These observations suggest that both sulphation and glucuronidation are critical determinants of the oral bioavailability of bioflavonoids in humans, although a contribution from CYP-mediated oxidation can not be excluded.

Bioactivation of the cooked food mutagen N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by estrogen sulfotransferase in cultured human mammary epithelial cells.

Cooked food mutagens from fried meat and fish have recently been suggested to contribute to the etiology of breast cancer. Thus, the most prevalent of these compounds, i.e. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, or rather its more mutagenic N-hydroxylated metabolite (N-OH-PhIP), forms DNA adducts in mammary cells, including human mammary epithelial (HME) cells. The objective of this study was to determine the involvement of estrogen sulfotransferase (EST), the only sulfotransferase identified in HME cells, in the further bioactivation of N-OH-PhIP. These studies were done in vitro using human recombinant EST and in intact HME cells. Human recombinant EST increased the covalent binding of [3H]N-OH-PhIP to calf thymus DNA approximately 3.5-fold in the presence of the sulfotransferase co-substrate 3'-phosphoadenosine-5'-phosphosulfate at each N-OH-PhIP concentration (1, 10 and 100 microM) (n = 6, P < 0.001). In contrast, EST did not catalyze the DNA binding of two other cooked food mutagens, N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, which are mainly hepatocarcinogens. Cultured HME cells displayed high EST activity, which could be completely inhibited by 1 microM estrone. When the cells were incubated with [3H]N-OH-PhIP, binding to native DNA occurred at 60-240 pmol/mg DNA. This binding was inhibited to 55% of control by 1 microM estrone (P < 0.01, n = 8), suggesting that EST plays a significant role in carcinogen bioactivation in human breast tissue.

Taxol transport by human intestinal epithelial Caco-2 cells.

Taxol (paclitaxel) belongs to a new class of antimicrotubule anticancer drugs with clinical activity against common solid tumors and acute leukemias. Preclinical studies have suggested that taxol is not absorbed after oral doses. However, whether the observed low oral bioavailability is the result of poor absorption or extensive presystemic hepatic metabolism is not clear. For this reason, we studied the transepithelial flux of taxol, using the human colonic cell line Caco-2 as a model. The cells were grown to confluency on permeable polycarbonate membrane inserts, to permit flux experiments after loading of [3H]taxol on either the apical or basolateral side. The flux of taxol across the Caco-2 cell layer was linear with time for up to 3 hr. The flux from the basolateral to the apical side was 4-10 times greater than that from the apical to the basolateral side. Whereas the absorptive transport appeared linearly related to the taxol concentration (0.5-20 microM), the efflux was saturable. The apparent KM of the active efflux component was 16.5 microM. Verapamil (50 microM) significantly decreased the active transport component. These data support the conclusion that rapid passive diffusion of taxol through the intestinal epithelium is partially counteracted by the action of an outwardly directed efflux pump, presumably P-glycoprotein. However, the relatively high apparent permeability coefficient for the apical to basolateral taxol transport (4.4 +/- 0.4 x 10(-6) cm/s; N = 17) suggests that the drug may still be effectively absorbed in the intestinal tract.

Extensive binding of the bioflavonoid quercetin to human plasma proteins.

Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma-protein binding of the most abundant bioflavonoid, quercetin, using 14C-labelled quercetin. An ultracentrifugation assay (170,000 g for 16 h at 20 degrees C) was shown to sediment plasma proteins. Binding of quercetin to normal plasma was extensive (99.1+/-0.5%, mean +/- s.d., n = 5). The unbound fraction varied as much as 6-fold, 0.3-1.8%, between subjects. This high binding was independent of quercetin concentration over the range 1.5-15 microM (0.5-5 microg mL(-1)). Human serum albumin was the primary protein responsible for the binding of quercetin in plasma (99.4+/-0.1%). Binding by alpha1-acid glycoprotein (39.2+/-0.5%) and very-low-density lipoproteins (<0.5% of total quercetin) did not make substantial contributions to overall plasma binding. The equilibrium association constant for the binding of quercetin to serum albumin was 267+/-33 x 10(3) M(-1) (n = 15). Thermodynamic data for the binding of quercetin to serum albumin indicated spontaneous, endothermic association. Displacement studies suggested that in man the 'IIA' subdomain binding site of human serum albumin was the primary binding site for quercetin. Association of quercetin with erythrocytes was significantly (P < 0.001) reduced by plasma protein binding. These data indicate poor cellular availability of quercetin because of its extensive binding to plasma proteins.